U.S. patent number 7,122,658 [Application Number 09/718,754] was granted by the patent office on 2006-10-17 for seed-preferred regulatory elements and uses thereof.
This patent grant is currently assigned to Pioneer Hi-Bred International, Inc.. Invention is credited to Shane E. Abbitt, William J. Gordon-Kamm, Kathryn K. Lappegard, Keith S. Lowe, Susan J. Martino-Catt, Jinrui Shi.
United States Patent |
7,122,658 |
Lappegard , et al. |
October 17, 2006 |
**Please see images for:
( Certificate of Correction ) ** |
Seed-preferred regulatory elements and uses thereof
Abstract
The present invention provides compositions and methods for
regulating expression of isolated nucleotide sequences in a plant.
The compositions are novel nucleic acid sequences for
seed-preferred regulatory sequences. Methods for expressing an
isolated nucleotide sequence in a plant using the regulatory
sequences are also provided. The methods comprise transforming a
plant cell to contain an isolated nucleotide sequence operably
linked to the seed-preferred regulatory sequences of the present
invention and regenerating a stably transformed plant from the
transformed plant cell.
Inventors: |
Lappegard; Kathryn K. (Nevada,
IA), Abbitt; Shane E. (Ankeny, IA), Martino-Catt; Susan
J. (Grimes, IA), Shi; Jinrui (Johnston, IA),
Gordon-Kamm; William J. (Urbandale, IA), Lowe; Keith S.
(Johnston, IA) |
Assignee: |
Pioneer Hi-Bred International,
Inc. (Johnston, IA)
|
Family
ID: |
24887377 |
Appl.
No.: |
09/718,754 |
Filed: |
November 22, 2000 |
Current U.S.
Class: |
536/24.1;
435/320.1 |
Current CPC
Class: |
C07K
14/415 (20130101); C12N 15/8234 (20130101); C12N
9/90 (20130101); C12Y 505/01004 (20130101) |
Current International
Class: |
C12N
15/29 (20060101); C12N 15/82 (20060101) |
Field of
Search: |
;800/287,278,320,320.1,298 ;435/69.1,320.1,419,468,412
;536/23.1,23.2,24.1,23.6 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
WO 98/37184 |
|
Aug 1998 |
|
WO |
|
WO 99/67405 |
|
Dec 1999 |
|
WO |
|
Other References
Lohmann et al., A Molecular Link between Stem Cell Regulation and
Floral Pattetning in Arabidopsis, Jun. 5, 2001, Cell, vol. 150, pp.
793-803. cited by examiner .
Busch et al., Activation of a Floral Homeotic Gene in Arabidopsis,
Jul. 23, 1999, Science, vol. 285, pp. 585-587. cited by examiner
.
Izawa et al., Plant bZIP Protein DNA Binding Specificity, 1993, J.
Mol. Biol., vol. 203, pp. 1131-1144. cited by examiner .
Hao et al., Unique Mode of GCC Box Recognition by the DNA-binding
Domain of . . . , Oct. 9, 1998, The Journal of Biological
Chemistry, vol. 273, No. 41, pp. 26857-26861. cited by examiner
.
Kagaya et al (1995, Mol. Gen. Genet. 248 :668-674). cited by
examiner .
Benfey et al (1990, Science 250:959-966). cited by examiner .
Benfey et al (1989, EMBO J, 8(8):2195-2202). cited by examiner
.
Ezcurra et al (1999, Plant Molecular Biology 40:699-709). cited by
examiner .
Kagaya et al (1995, Mol. Gen. Genet. 248 :668-674). cited by
examiner .
Benfey et al (1990, Science 250:959-966). cited by examiner .
Benfey et al (1989, EMBO J, 8(8):2195-2202). cited by examiner
.
Johnson, M.D., "The Arabidopsis thaliana myo-Inositol 1-Phosphate
Synthase (EC 5.5.1.4).sup.1", Plant Physiol 105:1023-1024 (1994).
cited by other .
Walbot, V., GenBank Accession No. AW066683, "Maize ESTs from
various cDNA libraries sequenced at Stanford University" (1999).
cited by other .
Kurek et al., Isolation and characterization of the wheat prolyl
isomerase FK506-binding protein (FKBP) 73 promoter, Plant Mol.
Biol. 42:489-497 (2000). cited by other .
Chen et al., Minimal regions in the Arabidopsis pistillata promoter
responsive to the Apetala3/Pistillata feedback control do not
contain a CArG box, Sex Plant Reprod. 13:85-94 (2000). cited by
other .
Ezcurra et al., Interaction between composite elements in the napA
promoter: both the B-box ABA-responsive complex and the RY/G
complex are necessary for seed-specific expression, Plant Mol.
Biol. 40:699-709 (1999). cited by other .
Wu et al., Quantitative nature of the Prolamin-box, ACGT and AACA
motifs in a rice glutelin gene promoter: minimal cis-element
requirements for endosperm-specific gene expression, Plant J.
23(3):415-421 (2000). cited by other .
Iida et al., Positive and negative cis-regulatory regions in the
soybean glycinin promoter identified by quantitative transient gene
expression, Plant Cell Reports (1995) 14:539-544. cited by other
.
Lee et al., Jasmonate signalling can be uncoupled from abscisic
acid signaling in barley: identification of jasmonate-regulated
transcripts which are not induced by abscisic acid, Planta (1996)
625-632. cited by other .
Lotan et al., Arabidopsis Leafy Cotyledon1 is Sufficient to Induce
Embryo Development in Vegetative Cells, Cell (1998) 93:1195-1205.
cited by other .
Smith et al., Temporal and spatial regulation of a novel gene in
barley embryos, Plant Molecular Biology (1992) 20:255-266. cited by
other .
Sundaresan et al., Patterns of gene action in plant development
revealed by enhancer trap and gene trap transposable elements,
Genes & Development (1995) 9:1797-1810. cited by other .
Walbot, V., 683006D10.x1 683--14 day immature embryo from Hake lab
(HS) Zea mays cDNA mRNA sequence, Database Accession No. AW066683
(1999). cited by other.
|
Primary Examiner: Baum; Stuart F.
Attorney, Agent or Firm: Pioneer Hi-Bred International, Inc.
Lappegard; Kathryn K.
Claims
What is claimed is:
1. An isolated promoter that drives transcription in a
seed-preferred manner, wherein said promoter comprises a nucleotide
sequence comprising the nucleotide sequence set forth in SEQ ID NO:
1.
2. An expression cassette comprising a promoter operably linked to
a nucleotide sequence wherein the promoter comprises the nucleotide
sequence set forth in SEQ ID NO:1.
Description
FIELD OF THE INVENTION
The present invention relates to the field of plant molecular
biology, more particularly to regulation of gene expression in
plants.
BACKGROUND OF THE INVENTION
Expression of isolated DNA sequences in a plant host is dependent
upon the presence of operably linked regulatory elements that are
functional within the plant host. Choice of the regulatory
sequences will determine when and where within the organism the
isolated DNA sequence is expressed. Where continuous expression is
desired throughout the cells of a plant, constitutive promoters are
utilized. In contrast, where gene expression in response to a
stimulus is desired, inducible promoters are the regulatory element
of choice. Where expression in specific tissues or organs are
desired, tissue-preferred promoters and/or terminators are used.
That is, these regulatory elements can drive expression in specific
tissues or organs. Additional regulatory sequences upstream and/or
downstream from the core sequences can be included in expression
cassettes of transformation vectors to bring about varying levels
of expression of isolated nucleotide sequences in a transgenic
plant.
Seed development involves embryogenesis and maturation events as
well as physiological adaptation processes that occur within the
seed to insure progeny survival. Developing plant seeds accumulate
and store carbohydrate, lipid, and protein that are subsequently
used during germination. Expression of storage protein genes in
seeds occurs primarily in the embryonic axis and cotyledons and in
the endosperm of developing seeds but never in mature vegetative
tissues. Generally, the expression patterns of seed proteins are
highly regulated. This regulation includes spatial and temporal
regulation during seed development. A variety of proteins
accumulate and decay during embryogenesis and seed development and
provide an excellent system for investigating different aspects of
gene regulation as well as for providing regulatory sequences for
use in genetic manipulation of plants.
Isolation and characterization of seed-preferred promoters and
terminators that can serve as regulatory elements for expression of
isolated nucleotide sequences of interest in a seed-preferred
manner are needed for improving seed traits in plants.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the invention, nucleotide sequences are provided
that allow initiation of transcription in seed. The sequences of
the invention comprise transcriptional initiation regions
associated with seed formation and seed tissues. Thus, the
compositions of the present invention comprise novel nucleotide
sequences for plant regulatory elements natively associated with
the nucleotide sequences coding for maize Jip1 (jasmonate-induced
protein), maize mi1ps3, (myo-inositol 1 phosphate synthase 3) and
maize Lec1 (leafy cotyledon 1).
A method for expressing an isolated nucleotide sequence in a plant
using the transcriptional initiation sequences disclosed herein is
provided. The method comprises transforming a plant cell with a
transformation vector that comprises an isolated nucleotide
sequence operably linked to one or more of the plant regulatory
sequences of the present invention and regenerating a stably
transformed plant from the transformed plant cell. In this manner,
the regulatory sequences are useful for controlling the expression
of endogenous as well as exogenous products in a seed-preferred
manner.
Under the transcriptional initiation regulation of the
seed-specific region will be a sequence of interest, which will
provide for modification of the phenotype of the seed. Such
modification includes modulating the production of an endogenous
product, as to amount, relative distribution, or the like, or
production of an exogenous expression product to provide for a
novel function or product in the seed.
By "seed-preferred" is intended favored expression in the seed,
including at least one of embryo, kernel, pericarp, endosperm,
nucellus, aleurone, pedicel, and the like.
By "regulatory element" is intended sequences responsible for
tissue and temporal expression of the associated coding sequence
including promoters, terminators, enhancers, introns, and the
like.
By "terminator" is intended sequences that are needed for
termination of transcription. A regulatory region of DNA that
causes RNA polymerase to disassociate from DNA, causing termination
of transcription.
By "promoter" is intended a regulatory region of DNA usually
comprising a TATA box capable of directing RNA polymerase II to
initiate RNA synthesis at the appropriate transcription initiation
site for a particular coding sequence. A promoter can additionally
comprise other recognition sequences generally positioned upstream
or 5' to the TATA box, referred to as upstream promoter elements,
which influence the transcription initiation rate. It is recognized
that having identified the nucleotide sequences for the promoter
region disclosed herein, it is within the state of the art to
isolate and identify further regulatory elements in the 5'
untranslated region upstream from the particular promoter region
identified herein. Thus the promoter region disclosed herein is
generally further defined by comprising upstream regulatory
elements such as those responsible for tissue and temporal
expression of the coding sequence, enhancers and the like. In the
same manner, the promoter elements which enable expression in the
desired tissue such as the seed can be identified, isolated, and
used with other core promoters to confirm seed-preferred
expression.
The isolated promoter sequences of the present invention can be
modified to provide for a range of expression levels of the
isolated nucleotide sequence. Less than the entire promoter region
can be utilized and the ability to drive seed-preferred expression
retained. However, it is recognized that expression levels of mRNA
can be decreased with deletions of portions of the promoter
sequence. Thus, the promoter can be modified to be a weak or strong
promoter. Generally, by "weak promoter" is intended a promoter that
drives expression of a coding sequence at a low level. By "low
level" is intended levels of about 1/10,000 transcripts to about
1/100,000 transcripts to about 1/500,000 transcripts. Conversely, a
strong promoter drives expression of a coding sequence at a high
level, or at about 1/10 transcripts to about 1/100 transcripts to
about 1/1,000 transcripts. Generally, at least about 20 nucleotides
of an isolated promoter sequence will be used to drive expression
of a nucleotide sequence.
It is recognized that to increase transcription levels enhancers
can be utilized in combination with the promoter regions of the
invention. Enhancers are nucleotide sequences that act to increase
the expression of a promoter region. Enhancers are known in the art
and include the SV40 enhancer region, the 35S enhancer element, and
the like.
The promoters of the present invention can be isolated from the 5'
untranslated region flanking its respective transcription
initiation site. Likewise the terminator can be isolated from the
3' untranslated region flanking its respective stop codon. The term
"isolated" refers to material, such as a nucleic acid or protein,
which is: (1) substantially or essentially free from components
which normally accompany or interact with the material as found in
its naturally occurring environment or (2) if the material is in
its natural environment, the material has been altered by
deliberate human intervention to a composition and/or placed at a
locus in a cell other than the locus native to the material.
Methods for isolation of promoter regions are well known in the
art. One method is described in U.S. patent application Ser. No.
06/098,690 filed Aug. 31, 1998 herein incorporated by reference.
The sequences for the promoter regions are set forth in SEQ ID NOS:
1, 4, and 7.
The Jip 1 promoter set forth in SEQ ID NO: 1 is 1,247 nucleotides
in length. A putative CAAT motif is found from position 861 864 and
a putative TATA motif is found from position 881 885. The promoter
was isolated from a coding sequence found in maize tissue libraries
of 11 to 30 DAP (days after pollination) endosperm and 13 to 40 DAP
embryo. The coding region has 40% homology to a barley
jasmonate-induced protein. The promoter can be isolated with the
primers of SEQ ID NOS: 2 and 3. The Jip 1 promoter can address
expression problems by providing expression throughout the whole
seed over a broad window of development.
The mi1 ps3 promoter set forth in SEQ ID NO: 4 is 752 nucleotides
in length. A putative CAAT motif is found from position 546 549 and
a putative TATA motif is found from position 576 580. The promoter
was isolated from a coding sequence found in maize tissue libraries
of 13 to 40 DAP embryo. The coding region has 94% homology to maize
mi1ps. The promoter can be isolated with the primers of SEQ ID NOS:
5 and 6. The mi1ps3 promoter can address expression problems by
directing expression preferentially to the embryo from mid-to-late
development.
The Lec1 promoter set forth in SEQ ID NO: 7 is 1,433 nucleotides in
length. A putative CAAT motif is found from position 836 839 and a
putative TATA motif is found from position 870 873. The promoter
was isolated from a coding sequence found in maize tissue libraries
of 13 to 20 DAP (days after pollination) embryo and callus tissue.
The coding region was identified according to the procedure
described in WO 00/28058 filed Nov. 9, 1998, incorporated herein by
reference. The promoter can be isolated with the primers/probes of
SEQ ID NOS: 8 and 9. The Lec 1 promoter can provide expression
during early embryo development and during the callus stage of
plant regeneration.
The promoter regions of the invention may be isolated from any
plant, including, but not limited to corn (Zea mays), canola
(Brassica napus, Brassica rapa ssp.), alfalfa (Medicago sativa),
rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum
bicolor, Sorghum vulgare), sunflower (Helianthus annuus), wheat
(Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana
tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea),
cotton (Gossypium hirsutum), sweet potato (Ipomoea batatus),
cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos
nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.),
cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa
spp.), avocado (Persea americana), fig (Ficus casica), guava
(Psidium guajava), mango (Mangifera indica), olive (Olea europaea),
oats, barley, vegetables, ornamentals, and conifers. Preferably,
plants include corn, soybean, sunflower, safflower, canola, wheat,
barley, rye, alfalfa, and sorghum.
Promoter sequences from other plants may be isolated according to
well-known techniques based on their sequence homology to the
promoter sequences set forth herein. In these techniques, all or
part of the known promoter sequence is used as a probe which
selectively hybridizes to other sequences present in a population
of cloned genomic DNA fragments (i.e. genomic libraries) from a
chosen organism. Methods are readily available in the art for the
hybridization of nucleic acid sequences.
The entire promoter sequence or portions thereof can be used as a
probe capable of specifically hybridizing to corresponding promoter
sequences. To achieve specific hybridization under a variety of
conditions, such probes include sequences that are unique and are
preferably at least about 10 nucleotides in length, and most
preferably at least about 20 nucleotides in length. Such probes can
be used to amplify corresponding promoter sequences from a chosen
organism by the well-known process of polymerase chain reaction
(PCR). This technique can be used to isolate additional promoter
sequences from a desired organism or as a diagnostic assay to
determine the presence of the promoter sequence in an organism.
Examples include hybridization screening of plated DNA libraries
(either plaques or colonies; see e.g. Innis et al. (1990) PCR
Protocols, A Guide to Methods and Applications, eds., Academic
Press).
The terms "stringent conditions" or "stringent hybridization
conditions" includes reference to conditions under which a probe
will hybridize to its target sequence, to a detectably greater
degree than other sequences (e.g., at least 2-fold over
background). Stringent conditions are target-sequence dependent and
will differ depending on the structure of the polynucleotide. By
controlling the stringency of the hybridization and/or washing
conditions, target sequences can be identified which are 100%
complementary to a probe (homologous probing). Alternatively,
stringency conditions can be adjusted to allow some mismatching in
sequences so that lower degrees of similarity are detected
(heterologous probing). Generally, probes of this type are in a
range of about 250 nucleotides in length to about 1000 nucleotides
in length.
An extensive guide to the hybridization of nucleic acids is found
in Tijssen, Laboratory Techniques in Biochemistry and Molecular
Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2
"Overview of principles of hybridization and the strategy of
nucleic acid probe assays", Elsevier, New York (1993); and Current
Protocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds.,
Greene Publishing and Wiley-Interscience, New York (1995). See also
Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd
ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
In general, sequences that correspond to the promoter sequence of
the present invention and hybridize to the promoter sequence
disclosed herein will be at least 50% homologous, 55% homologous,
60% homologous, 65% homologous, 70% homologous, 75% homologous, 80%
homologous, 85% homologous, 90% homologous, 95% homologous and even
98% homologous or more with the disclosed sequence.
Specificity is typically the function of post-hybridization washes,
the critical factors being the ionic strength and temperature of
the final wash solution. Generally, stringent wash temperature
conditions are selected to be about 5.degree. C. to about 2.degree.
C. lower than the melting point (T.sub.m) for the specific sequence
at a defined ionic strength and pH. The melting point, or
denaturation, of DNA occurs over a narrow temperature range and
represents the disruption of the double helix into its
complementary single strands. The process is described by the
temperature of the midpoint of transition, T.sub.m, which is also
called the melting temperature. Formulas are available in the art
for the determination of melting temperatures.
Hybridization conditions for the promoter sequences of the
invention include hybridization at 42.degree. C. in 50% (w/v)
formamide, 6.times.SSC, 0.5% (w/v) SDS, 100 .mu.g/ml salmon sperm
DNA. Exemplary low stringency washing conditions include
hybridization at 42.degree. C. in a solution of 2.times.SSC, 0.5%
(w/v) SDS for 30 minutes and repeating. Exemplary moderate
stringency conditions include a wash in 2.times.SSC, 0.5% (w/v) SDS
at 50.degree. C. for 30 minutes and repeating. Exemplary high
stringency conditions include a wash in 2.times.SSC, 0.5% (w/v)
SDS, at 65.degree. C. for 30 minutes and repeating. Sequences that
correspond to the promoter of the present invention may be obtained
using all the above conditions.
The following terms are used to describe the sequence relationships
between two or more nucleic acids or polynucleotides: (a)
"reference sequence", (b) "comparison window", (c) "percentage of
sequence identity", and (d) "substantial identity".
(a) As used herein, "reference sequence" is a defined sequence used
as a basis for sequence comparison. A reference sequence may be a
subset or the entirety of a specified sequence; for example, as a
segment of a full-length promoter sequence, or the complete
promoter sequence.
(b) As used herein, "comparison window" makes reference to a
contiguous and specified segment of a polynucleotide sequence,
wherein the polynucleotide sequence may be compared to a reference
sequence and wherein the portion of the polynucleotide sequence in
the comparison window may comprise additions or deletions (i.e.,
gaps) compared to the reference sequence (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
Generally, the comparison window is at least 20 contiguous
nucleotides in length and optionally can be 30, 40, 50, 100, or
more contiguous nucleotides in length. Those of skill in the art
understand that to avoid a high similarity to a reference sequence
due to inclusion of gaps in the polynucleotide sequence a gap
penalty is typically introduced and is subtracted from the number
of matches.
(c) As used herein, "percentage of sequence identity" means the
value determined by comparing two optimally aligned sequences over
a comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base occurs
in both sequences to yield the number of matched positions,
dividing the number of matched positions by the total number of
positions in the window of comparison and multiplying the result by
100 to yield the percentage of sequence identity.
(d) The term "substantial identity" of polynucleotide sequences
means that a polynucleotide comprises a sequence that has at least
70% sequence identity, preferably at least 80%, more preferably at
least 90% and most preferably at least 95%, compared to a reference
sequence using one of the alignment programs described using
standard parameters.
Methods of aligning sequences for comparison are well known in the
art. Gene comparisons can be determined by conducting BLAST (Basic
Local Alignment Search Tool; Altschul, S. F., et al., (1993) J.
Mol. Biol. 215:403 410 searches under default parameters for
identity to sequences contained in the BLAST "GENEMBL" database. A
sequence can be analyzed for identity to all publicly available DNA
sequences contained in the GENEMBL database using the BLASTN
algorithm under the default parameters. Identity to the sequence of
the present invention would mean a polynucleotide sequence having
at least 65% sequence identity, more preferably at least 70%
sequence identity, more preferably at least 75% sequence identity,
more preferably at least 80% identity, more preferably at least 85%
sequence identity, more preferably at least 90% sequence identity
and most preferably at least 95% sequence identity wherein the
percent sequence identity is based on the entire promoter
region.
GAP uses the algorithm of Needleman and Wunsch (J. Mol. Biol.
48:443 453, 1970) to find the alignment of two complete sequences
that maximizes the number of matches and minimizes the number of
gaps. GAP considers all possible alignments and gap positions and
creates the alignment with the largest number of matched bases and
the fewest gaps. It allows for the provision of a gap creation
penalty and a gap extension penalty in units of matched bases. GAP
must make a profit of gap creation penalty number of matches for
each gap it inserts. If a gap extension penalty greater than zero
is chosen, GAP must, in addition, make a profit for each gap
inserted of the length of the gap times the gap extension penalty.
Default gap creation penalty values and gap extension penalty
values in Version 10 of the Wisconsin Genetics Software Package for
protein sequences are 8 and 2, respectively. For nucleotide
sequences the default gap creation penalty is 50 while the default
gap extension penalty is 3. The gap creation and gap extension
penalties can be expressed as an integer selected from the group of
integers consisting of from 0 to 200. Thus, for example, the gap
creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or
greater.
GAP presents one member of the family of best alignments. There may
be many members of this family, but no other member has a better
quality. GAP displays four figures of merit for alignments:
Quality, Ratio, Identity, and Similarity. The Quality is the metric
maximized in order to align the sequences. Ratio is the quality
divided by the number of bases in the shorter segment. Percent
Identity is the percent of the symbols that actually match. Percent
Similarity is the percent of the symbols that are similar. Symbols
that are across from gaps are ignored. A similarity is scored when
the scoring matrix value for a pair of symbols is greater than or
equal to 0.50, the similarity threshold. The scoring matrix used in
Version 10 of the Wisconsin Genetics Software Package is BLOSUM62
(see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA
89:10915).
Sequence fragments with high percent identity to the sequences of
the present invention also refer to those fragments of a particular
regulatory element nucleotide sequence disclosed herein that
operate to promote the seed-preferred expression of an operably
linked isolated nucleotide sequence. These fragments will comprise
at least about 20 contiguous nucleotides, preferably at least about
50 contiguous nucleotides, more preferably at least about 75
contiguous nucleotides, even more preferably at least about 100
contiguous nucleotides of the particular promoter nucleotide
sequence disclosed herein. The nucleotides of such fragments will
usually comprise the TATA recognition sequence of the particular
promoter sequence. Such fragments can be obtained by use of
restriction enzymes to cleave the naturally occurring regulatory
element nucleotide sequences disclosed herein; by synthesizing a
nucleotide sequence from the naturally occurring DNA sequence; or
can be obtained through the use of PCR technology. See
particularly, Mullis et al. (1987) Methods Enzymol. 155:335 350,
and Erlich, ed. (1989) PCR Technology (Stockton Press, New York).
Again, variants of these fragments, such as those resulting from
site-directed mutagenesis, are encompassed by the compositions of
the present invention.
Nucleotide sequences comprising at least about 20 contiguous
sequences of the sequence set forth in SEQ ID NOS:1, 4, 7, or 10
are encompassed. These sequences can be isolated by hybridization,
PCR, and the like. Such sequences encompass fragments capable of
driving seed-preferred expression, fragments useful as probes to
identify similar sequences, as well as elements responsible for
temporal or tissue specificity.
Biologically active variants of the regulatory sequences are also
encompassed by the compositions of the present invention. A
regulatory "variant" is a modified form of a regulatory sequence
wherein one or more bases have been modified, removed or added. For
example, a routine way to remove part of a DNA sequence is to use
an exonuclease in combination with DNA amplification to produce
unidirectional nested deletions of double stranded DNA clones. A
commercial kit for this purpose is sold under the trade name
Exo-Size.TM. (New England Biolabs, Beverly, Mass.). Briefly, this
procedure entails incubating exonuclease III with DNA to
progressively remove nucleotides in the 3' to 5' direction at 5'
overhangs, blunt ends or nicks in the DNA template. However,
exonuclease III is unable to remove nucleotides at 3', 4-base
overhangs. Timed digests of a clone with this enzyme produces
unidirectional nested deletions.
One example of a regulatory sequence variant is a promoter formed
by one or more deletions from a larger promoter. The 5' portion of
a promoter up to the TATA box near the transcription start site can
be deleted without abolishing promoter activity, as described by
Zhu et al., The Plant Cell 7: 1681 89 (1995). Such variants should
retain promoter activity, particularly the ability to drive
expression in seed or seed tissues. Biologically active variants
include, for example, the native regulatory sequences of the
invention having one or more nucleotide substitutions, deletions or
insertions. Activity can be measured by Northern blot analysis,
reporter activity measurements when using transcriptional fusions,
and the like. See, for example, Sambrook et al. (1989) Molecular
Cloning: A Laboratory Manual (2nd ed. Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.), herein incorporated by
reference.
The nucleotide sequences for the seed-preferred regulatory elements
disclosed in the present invention, as well as variants and
fragment thereof, are useful in the genetic manipulation of any
plant when operably linked with an isolated nucleotide sequence
whose expression is to be controlled to achieve a desired
phenotypic response. By "operably linked" is intended the
transcription or translation of the isolated nucleotide sequence is
under the influence of the regulatory sequence. In this manner, the
nucleotide sequences for the regulatory elements of the invention
may be provided in expression cassettes along with isolated
nucleotide sequences for expression in the plant of interest, more
particularly in the seed of the plant. Such an expression cassette
is provided with a plurality of restriction sites for insertion of
the nucleotide sequence to be under the transcriptional control of
the regulatory elements.
The genes of interest expressed by the regulatory elements of the
invention can be used for varying the phenotype of seeds. This can
be achieved by increasing expression of endogenous or exogenous
products in seeds. Alternatively, the results can be achieved by
providing for a reduction of expression of one or more endogenous
products, particularly enzymes or cofactors in the seed. These
modifications result in a change in phenotype of the transformed
seed. It is recognized that the regulatory elements may be used
with their native coding sequences to increase or decrease
expression resulting in a change in phenotype in the transformed
seed.
In another embodiment, the regulatory elements of the invention can
be used for callus-preferred expression of selectable markers. For
example, regulatory elements such as the Lec1 promoter and
terminator would allow plants to be regenerated that have no field
resistance to herbicide but may be completely susceptible to the
herbicide in the callus stage.
General categories of genes of interest for the purposes of the
present invention include for example, those genes involved in
information, such as Zinc fingers; those involved in communication,
such as kinases; and those involved in housekeeping, such as heat
shock proteins. More specific categories of transgenes include
genes encoding important traits for agronomics, insect resistance,
disease resistance, herbicide resistance, and grain
characteristics. Still other categories of transgenes include genes
for inducing expression of exogenous products such as enzymes,
cofactors, and hormones from plants and other eukaryotes as well as
prokaryotic organisms. It is recognized that any gene of interest,
including the native coding sequence, can be operably linked to the
regulatory elements of the invention and expressed in the seed.
Modifications that affect grain traits include increasing the
content of oleic acid, or altering levels of saturated and
unsaturated fatty acids. Likewise, increasing the levels of lysine
and sulfur-containing amino acids may be desired as well as the
modification of starch type and content in the seed. Hordothionin
protein modifications are described in WO 9416078 filed Apr. 10,
1997; WO 9638562 filed Mar. 26, 1997; WO 9638563 filed Mar. 26,
1997 and U.S. Pat. No. 5,703,409 issued Dec. 30, 1997; the
disclosures of which are incorporated herein by reference. Another
example is lysine and/or sulfur-rich seed protein encoded by the
soybean 2S albumin described in WO 9735023 filed Mar. 20, 1996, and
the chymotrypsin inhibitor from barley, Williamson et al. (1987)
Eur. J. Biochem. 165:99 106, the disclosures of each are
incorporated by reference.
Derivatives of the following genes can be made by site-directed
mutagenesis to increase the level of preselected amino acids in the
encoded polypeptide. For example, the gene encoding the barley high
lysine polypeptide (BHL), is derived from barley chymotrypsin
inhibitor, WO 9820133 filed Nov. 1, 1996 the disclosure of which is
incorporated herein by reference. Other proteins include
methionine-rich plant proteins such as from sunflower seed, Lilley
et al. (1989) Proceedings of the World Congress on Vegetable
Protein Utilization in Human Foods and Animal Feedstuffs;
Applewhite, H. (ed.); American Oil Chemists Soc., Champaign, Ill.:
497 502, incorporated herein by reference; corn, Pedersen et al.
(1986) J. Biol. Chem. 261:6279; Kirihara et al. (1988) Gene 71:359,
both incorporated herein by reference; and rice, Musumura et al.
(1989) Plant Mol. Biol. 12:123, incorporated herein by reference.
Other important genes encode glucans, Floury 2, growth factors,
seed storage factors and transcription factors.
Agronomic traits in seeds can be improved by altering expression of
genes that: affect the response of seed growth and development
during environmental stress, Cheikh-N et al (1994) Plant Physiol.
106(1):45 51) and genes controlling carbohydrate metabolism to
reduce kernel abortion in maize, Zinselmeier et al. (1995) Plant
Physiol. 107(2):385 391.
Insect resistance genes may encode resistance to pests that have
great yield drag such as rootworm, cutworm, European Corn Borer,
and the like. Such genes include, for example: Bacillus
thuringiensis endotoxin genes, U.S. Pat. Nos. 5,366,892; 5,747,450;
5,737,514; 5,723,756; 5,593,881; Geiser et al. (1986) Gene 48:109;
lectins, Van Damme et al. (1994) Plant Mol. Biol. 24:825; and the
like.
Genes encoding disease resistance traits include: detoxification
genes, such as against fumonosin (WO 9606175 filed Jun. 7, 1995);
avirulence (avr) and disease resistance (R) genes, Jones et al.
(1994) Science 266:789; Martin et al. (1993) Science 262:1432;
Mindrinos et al. (1994) Cell 78:1089; and the like.
Commercial traits can also be encoded on a gene(s) which could
alter or increase for example, starch for the production of paper,
textiles and ethanol, or provide expression of proteins with other
commercial uses. Another important commercial use of transformed
plants is the production of polymers and bioplastics such as
described in U.S. Pat. No. 5,602,321 issued Feb. 11, 1997. Genes
such as B-Ketothiolase, PHBase (polyhydroxyburyrate synthase) and
acetoacetyl-CoA reductase (see Schubert et al. (1988) J. Bacteriol
170(12):5837 5847) facilitate expression of polyhyroxyalkanoates
(PHAs).
Exogenous products include plant enzymes and products as well as
those from other sources including prokaryotes and other
eukaryotes. Such products include enzymes, cofactors, hormones, and
the like. The level of seed proteins, particularly modified seed
proteins having improved amino acid distribution to improve the
nutrient value of the seed can be increased. This is achieved by
the expression of such proteins having enhanced amino acid
content.
The nucleotide sequence operably linked to the regulatory elements
disclosed herein can be an antisense sequence for a targeted gene.
By "antisense DNA nucleotide sequence" is intended a sequence that
is in inverse orientation to the 5'-to-3' normal orientation of
that nucleotide sequence. When delivered into a plant cell,
expression of the antisense DNA sequence prevents normal expression
of the DNA nucleotide sequence for the targeted gene. The antisense
nucleotide sequence encodes an RNA transcript that is complementary
to and capable of hybridizing with the endogenous messenger RNA
(mRNA) produced by transcription of the DNA nucleotide sequence for
the targeted gene. In this case, production of the native protein
encoded by the targeted gene is inhibited to achieve a desired
phenotypic response. Thus the regulatory sequences disclosed herein
can be operably linked to antisense DNA sequences to reduce or
inhibit expression of a native protein in the plant seed.
The expression cassette will also include at the 3' terminus of the
isolated nucleotide sequence of interest, a transcriptional and
translational termination region functional in plants. The
termination region can be native with the promoter nucleotide
sequence of the present invention, can be native with the DNA
sequence of interest, or can be derived from another source.
The Lec1 terminator set forth in SEQ ID NO: 10 is 695 nucleotides
in length. The terminator was isolated from a coding sequence found
in maize tissue libraries of 13 to 20 DAP (days after pollination)
embryo and callus tissue. The coding region was identified
according to the procedure described in WO 00/28058 filed Nov. 9,
1998, incorporated herein by reference. The terminator can be
isolated with the primers/probes of SEQ ID NOS: 11 and 12. The Lec
1 terminator, with the appropriate promoter, can provide expression
during early embryo development and during the callus stage of
plant regeneration. The Lec1 terminator can be used with the Lec1
promoter in an expression cassette, or can be used with another
appropriate promoter to provide seed-preferred or callus-preferred
expression of a coding region.
Other convenient termination regions are available from the
Ti-plasmid of A. tumefaciens, such as the octopine synthase and
nopaline synthase termination regions. See also: Guerineau et al.
(1991) Mol. Gen. Genet. 262:141 144; Proudfoot (1991) Cell 64:671
674; Sanfacon et al. (1991) Genes Dev. 5:141 149; Mogen et al.
(1990) Plant Cell 2:1261 1272; Munroe et al. (1990) Gene 91:151
158; Ballas et al. 1989) Nucleic Acids Res. 17:7891 7903; Joshi et
al. (1987) Nucleic Acid Res. 15:9627 9639.
The expression cassettes can additionally contain 5' leader
sequences. Such leader sequences can act to enhance translation.
Translation leaders are known in the art and include: picornavirus
leaders, for example: EMCV leader (Encephalomyocarditis 5'
noncoding region), Elroy-Stein et al. (1989) Proc. Nat. Acad. Sci.
USA 86:6126 6130; potyvirus leaders, for example, TEV leader
(Tobacco Etch Virus), Allison et al. (1986); MDMV leader (Maize
Dwarf Mosaic Virus), Virology 154:9 20; human immunoglobulin
heavy-chain binding protein (BiP), Macejak et al. (1991) Nature
353:90 94; untranslated leader from the coat protein mRNA of
alfalfa mosaic virus (AMV RNA 4), Jobling et al. (1987) Nature
325:622 625); tobacco mosaic virus leader (TMV), Gallie et al.
(1989) Molecular Biology of RNA, pages 237 256; and maize chlorotic
mottle virus leader (MCMV) Lommel et al. (1991) Virology 81:382
385. See also Della-Cioppa et al. (1987) Plant Physiology 84:965
968. The cassette can also contain sequences that enhance
translation and/or mRNA stability such as introns.
In those instances where it is desirable to have the expressed
product of the isolated nucleotide sequence directed to a
particular organelle, particularly the plastid, amyloplast, or to
the endoplasmic reticulum, or secreted at the cell's surface or
extracellularly, the expression cassette can further comprise a
coding sequence for a transit peptide. Such transit peptides are
well known in the art and include, but are not limited to: the
transit peptide for the acyl carrier protein, the small subunit of
RUBISCO, plant EPSP synthase, and the like.
In preparing the expression cassette, the various DNA fragments can
be manipulated, so as to provide for the DNA sequences in the
proper orientation and, as appropriate, in the proper reading
frame. Toward this end, adapters or linkers can be employed to join
the DNA fragments or other manipulations can be involved to provide
for convenient restriction sites, removal of superfluous DNA,
removal of restriction sites, or the like. For this purpose, in
vitro mutagenesis, primer repair, restriction digests, annealing,
and resubstitutions such as transitions and transversions, can be
involved.
As noted herein, the present invention provides vectors capable of
expressing genes of interest under the control of the regulatory
elements. In general, the vectors should be functional in plant
cells. At times, it may be preferable to have vectors that are
functional in E. coli (e.g., production of protein for raising
antibodies, DNA sequence analysis, construction of inserts,
obtaining quantities of nucleic acids). Vectors and procedures for
cloning and expression in E. coli are discussed in Sambrook et al.
(supra).
The transformation vector comprising the regulatory sequences of
the present invention operably linked to an isolated nucleotide
sequence in an expression cassette, can also contain at least one
additional nucleotide sequence for a gene to be cotransformed into
the organism. Alternatively, the additional sequence(s) can be
provided on another transformation vector.
Vectors that are functional in plants can be binary plasmids
derived from Agrobacterium. Such vectors are capable of
transforming plant cells. These vectors contain left and right
border sequences that are required for integration into the host
(plant) chromosome. At minimum, between these border sequences is
the gene to be expressed under control of the regulatory elements
of the present invention. In one embodiment, a selectable marker
and a reporter gene are also included. For ease of obtaining
sufficient quantities of vector, a bacterial origin that allows
replication in E. coli can be used.
Reporter genes can be included in the transformation vectors.
Examples of suitable reporter genes known in the art can be found
in, for example: Jefferson et al. (1991) in Plant Molecular Biology
Manual, ed. Gelvin et al. (Kluwer Academic Publishers), pp. 1 33;
DeWet et al., (1987) Mol. Cell. Biol. 7:725 737; Goff et al. (1990)
EMBO J. 9:2517 2522; Kain et al., (1995) BioTechniques 19:650 655;
and Chiu et al. (1996) Current Biology 6:325 330.
Selectable marker genes for selection of transformed cells or
tissues can be included in the transformation vectors. These can
include genes that confer antibiotic resistance or resistance to
herbicides. Examples of suitable selectable marker genes include,
but are not limited to: genes encoding resistance to
chloramphenicol, Herrera Estrella et al., (1983) EMBO J. 2:987 992;
methotrexate, Herrera Estrella et al. (1983) Nature 303:209 213;
Meijer et al. (1991) Plant Mol. Biol. 16:807 820; hygromycin,
Waldron et al. (1985) Plant Mol. Biol. 5:103 108; Zhijian et al.
(1995) Plant Science 108:219 227; streptomycin, Jones et al.,
(1987) Mol. Gen. Genet. 210:86 91; spectinomycin, Bretagne-Sagnard
et al. (1996) Transgenic Res. 5:131 137; bleomycin, Hille et al.
(1990) Plant Mol. Biol. 7:171 176; sulfonamide, Guerineau et al.
(1990) Plant Mol. Biol. 15:127 136; bromoxynil, Stalker et al.
(1988) Science 242:419 423; glyphosate, Shaw et al. (1986) Science
233:478 481; phosphinothricin, DeBlock et al. (1987) EMBO J. 6:2513
2518.
Other genes that could serve utility in the recovery of transgenic
events but might not be required in the final product would
include, but are not limited to: GUS (.beta.-glucoronidase),
Jefferson (1987) Plant Mol. Biol. Rep. 5:387); GFP (green
florescence protein), Chalfie et al. (1994) Science 263:802;
luciferase, Teeri et al. (1989) EMBO J. 8:343; and the maize genes
encoding for anthocyanin production, Ludwig et al. (1990) Science
247:449.
The transformation vector comprising the particular regulatory
sequences of the present invention, operably linked to an isolated
nucleotide sequence of interest in an expression cassette, can be
used to transform any plant. In this manner, genetically modified
plants, plant cells, plant tissue, seed, and the like can be
obtained. Transformation protocols can vary depending on the type
of plant or plant cell, i.e., monocot or dicot, targeted for
transformation. Suitable methods of transforming plant cells
include microinjection, Crossway et al. (1986) Biotechniques 4:320
334; electroporation, Riggs et al. (1986) Proc. Natl. Acad. Sci.
USA 83:5602 5606; Agrobacteium-mediated transformation, see for
example, Townsend et al. U.S. Pat. No. 5,563,055; direct gene
transfer, Paszkowski et al. (1984) EMBO J. 3:2717 2722; and
ballistic particle acceleration, see for example, Sanford et al.
U.S. Pat. No. 4,945,050; Tomes et al. (1995) in Plant Cell, Tissue,
and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips
(Springer-Verlag, Berlin); and McCabe et al. (1988) Biotechnology
6:923 926. Also see Weissinger et al. (1988) Annual Rev. Genet.
22:421 477; Sanford et al. (1987) Particulate Science and
Technology 5:27 37 (onion); Christou et al. (1988) Plant Physiol.
87:671 674 (soybean); McCabe et al. (1988) Bio/Technology 6:923 926
(soybean); Datta et al. (1990) Biotechnology 8:736 740 (rice);
Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305 4309
(maize); Klein et al. (1988) Biotechnology 6:559 563 (maize); Klein
et al. (1988) Plant Physiol. 91:440 444 (maize); Fromm et al.
(1990) Biotechnology 8:833 839; Hooydaas-Van Slogteren et al.
(1984) Nature (London) 311:763 764; Bytebier et al. (1987) Proc.
Natl. Acad. Sci. USA 84:5345 5349 (Liliaceae); De Wet et al. (1985)
in The Experimental Manipulation of Ovule Tissues, ed. G. P.
Chapman et al. (Longman, New York), pp. 197 209 (pollen); Kaeppler
et al. (1990) Plant Cell Reports 9:415 418; and Kaeppler et al.
(1992) Theor. Appl. Genet. 84:560 566 (whisker-mediated
transformation); D. Halluin et al. (1992) Plant Cell 4:1495 1505
(electroporation); Li et al. (1993) Plant Cell Reports 12:250 255
and Christou et al. (1995) Annals of Botany 75:407 413 (rice);
Osjoda et al. (1996) Nature Biotechnology 14:745 750 (maize via
Agrobacterium tumefaciens); all of which are herein incorporated by
reference.
The cells that have been transformed can be grown into plants in
accordance with conventional ways. See, for example, McCormick et
al. (1986) Plant Cell Reports 5:81 84. These plants can then be
grown and pollinated with the same transformed strain or different
strains. The resulting hybrid having seed-preferred expression of
the desired phenotypic characteristic can then be identified. Two
or more generations can be grown to ensure that seed-preferred
expression of the desired phenotypic characteristic is stably
maintained and inherited.
The following examples are offered by way of illustration and not
by way of imitation.
EXAMPLES
The regulatory regions of the invention were isolated from maize
plants and cloned. The genes were selected as a source for a
seed-preferred regulatory regions based on the spatial expression
of their gene products. The method for their isolation is described
below.
Example 1
Isolation of Promoter Sequences Using Genome Walker
The procedure for promoter isolation is described in the User
Manual for the Genome Walker kit sold by Clontech Laboratories,
Inc., Palo Alto, Calif. Genomic DNA from maize line V3 4 A63 was
prepared by grinding 10-day-old seedling leaves in liquid nitrogen,
and the DNA prepared as described by Chen and Dellaporta (1994) in
The Maize Handbook, ed. Freeling and Walbot (Springer-Verlag,
Berlin) with a few minor modifications as follows: precipitated DNA
was recovered using an inoculation loop and transferred to a 1.5 ml
eppendorf tube containing 500 l of TE (10 mM Tris pH 8.0, 1 mM
EDTA). The DNA was allowed to dissolve at room temperature for 15
minutes, phenol extracted and 2-propanol precipitated in 700 l. The
precipitate was recovered and washed with 70% ethanol. The DNA was
then placed in a clean 1.5 ml eppendorf tube to air dry and
resuspended in 200 .mu.l of TE. RNase A was added to 10 .mu.g/ml
and the mixture was incubated at 37.degree. C. for several hours.
The DNA was then extracted once with phenol-chloroform, then
chloroform, then ethanol precipitated and resuspended in TE. The
DNA was then used as described in the Genome Walker User Manual
(Clontech PT3042-1 version PR68687) with the following
modifications: briefly, the DNA was digested separately with
restriction enzymes DraI, EcoRV, PvuII, ScaI, StuI, HpaI, EcoICRI,
XmnI, and SspI all blunt-end cutters. The DNA was extracted with
phenol, then chloroform, then ethanol precipitated. The Genome
Walker adapters were ligated onto the ends of the restricted DNA.
The resulting DNA is referred to as DL1 DL9, respectively.
For isolation of Jip1 and mi1ps3 promoter regions, two
nonoverlapping gene-specific primers (26 30 bp in length) were
designed from the 5' end of the maize genes identified from
sequence databases. The primers were designed to amplify the region
upstream of the coding sequence, i.e. the 5' untranslated region
and promoter of the chosen gene. The first round of PCR was
performed on each DNA sample (DL1 9) with Clontech primer AP1 and
the gene-specific primers (gsp)1 with the sequences shown in SEQ ID
NOS: 2 and 5.
PCR was performed in a Bio-Rad icycler (Hercules, Calif.) thermal
cycler using reagents supplied with the Genome Walker kit. The
following cycle parameters were used: 7 cycles of 94.degree. C. for
2 seconds, then 70.degree. C. for 3 minutes, followed by 36 cycles
of 94.degree. C. for 2 seconds and 67.degree. C. for 3 minutes.
Finally, the samples were held at 67.degree. C. for 4 minutes and
then at 4.degree. C. until further analysis.
For the mi1 ps3 promoter, bands from the primary PCR (DL-1, DL-7,
DL-8, and DL-9) were used to isolate the promoter sequence.
For the Jip1 promoter, as described in the User Manual, the DNA
from the first round of PCR was then diluted and used as a template
in a second round of PCR using the Clontech AP2 primer and
gene-specific primers (gsp2) with the sequence shown in SEQ ID NO:
3.
The cycle parameters for the second round were: 5 cycles of
94.degree. C. for 2 seconds, then 70.degree. C. for 3 minutes,
followed by 25 cycles of 94.degree. C. for 2 seconds and 67.degree.
C. for 3 minutes. Finally, the samples were held at 67.degree. C.
for 4 minutes and then held at 4.degree. C. Approximately 10 .mu.l
of each reaction were run on a 0.8% agarose gel, and bands (usually
500 bp or larger) were excised, purified with the Qiagen Gel
Extraction Kit (Santa Clarita, Calif.) and cloned into the pGEM-T
Easy vector (Promega Corp. Madison, Wis.). Clones were sequenced
for verification.
After verification, the promoters were reisolated by amplifying new
fragments from genomic DNA from maize line V3 4 A63 using primers
created from the new promoter sequence. This was done to insure
that sequences were error free. The PCR reaction was performed in a
Bio-Rad icycler (Hercules, Calif.) thermal cycler using Hifidelity
supermix (Cat.# 10790-020, Life Technologies, Rockville Md.). The
following cycle parameters were used: 94.degree. C. for 2 seconds,
followed by 30 cycles of 94.degree. C. for 20 seconds, (55.degree.
C. for Jip1, 61.degree. C. for mi1ps3) for 30 seconds, and
68.degree. C. for 1 minute. Finally, the samples were held at
67.degree. C. for 4 minutes and then at 4.degree. C. until further
analysis. The primers also contained sequences that would generate
an NcoI site at the start codon. This was performed in order to
create a translational fusion. For Jip1, these primers are shown in
SEQ ID NOS: 13 and 14, for mi1ps3, they are shown in SEQ ID NOS: 15
and 16.
Example 2
Isolation of Promoter Sequences Using BAC (Bacterial Artificial
Chromosome) Libraries
The BAC library subjected to the screening procedure was produced
from the maize Mo17 genotype using the protocol of Gubler and
Hoffman (Gubler, U. et al. Gene, 1983; 25:263 269). Half of the
library was digested with HindIII and half with EcoRI. The half cut
with HindIII and screened for the Lec1 promoter consisted of
approximately 69,000 clones. These clones were plated out on
22.times.22 cm2 agar plate at density of about 3,000 colonies per
plate. The plates were incubated in a 37.degree. C. incubator for
12 24 hours. Colonies were picked into 384-well plates by a robot
colony picker, Q-bot (GENETIX Limited). These plates were incubated
overnight at 37.degree. C.
Once sufficient colonies were picked, they were pinned onto
22.times.22 cm2 nylon membranes using Q-bot. Each membrane
contained 9,216 colonies or 36,864 colonies. These membranes were
placed onto agar plate with appropriate antibiotic. The plates were
incubated at 37.degree. C. for overnight.
After colonies were recovered on the second day, these filters were
placed on filter paper prewetted with denaturing solution for four
minutes, then were incubated on top of a boiling water bath for
additional four minutes. The filters were then placed on filter
paper prewetted with neutralizing solution for four minutes. After
excess solution was removed by placing the filters on dry filter
papers for one minute, the colony side of the filters were place
into Proteinase K solution, incubated at 37.degree. C. for 40 50
minutes. The filters were placed on dry filter papers to dry
overnight. DNA was then cross-linked to nylon membrane by UV light
treatment.
Colony hybridization was conducted as described by Sambrook, J.,
Fritsch, E. F. and Maniatis, T., (in Molecular Cloning: A
laboratory Manual, 2nd Edition). The probe was used in colony
hybridization. The probe was created in a PCR reaction using the
Advantage GC genomic PCR Kit (Clontech, Palo alto, CA) with 5% GC
melt. Genomic DNA from maize line V3 4 A63 was amplified with a
forward and reverse primer: SEQ ID NOS: 8, and 9. The PCR reaction
was performed in a Bio-Rad icycler (Hercules, Calif.) thermal
cycler using Hifidelity supermix (Cat.# 10790-020, Life
Technologies, Rockville Md.). The following cycle parameters were
used: 94.degree. C. for 2 seconds, followed by 30 cycles of
94.degree. C. for 20 seconds, 63.degree. C. for 30 seconds, and
68.degree. C. for 1 minute. Finally, the samples were held at
67.degree. C. for 4 minutes and then at 4.degree. C. until further
analysis.
Once positive BACs were identified, a southern analysis using a
HindIII digestion was performed on them to identify bands
containing Lec1 for subcloning. The same probe was used to identify
a fragment (.about.4 kb) which was positive for Lec1. This band was
subcloned into the pBS2KS+ cloning vector (Stratagene Inc., 11011
N. Torrey Pines Rd., La Jolla, Calif.) and sequenced. The band
contained 1.4 kb of sequence upstream of the coding region of Lec1
and 1.7 kb of sequence downstream. The Lec1 promoter and terminator
regions were obtained using primers SEQ ID NOS: 17, 18, 19 and 20,
created from this sequence to amplify genomic DNA from maize line
A63. The PCR reaction was performed in a Bio-Rad icycler (Hercules,
Calif.) thermal cycler using Hifidelity supermix (Cat.# 10790-020,
Life Technologies, Rockville Md.). The following cycle parameters
were used: 94.degree. C. for 2 seconds, followed by 30 cycles of
94.degree. C. for 20 seconds, 58.degree. C. for 30 seconds, and
68.degree. C. for 1 minute. Finally, the samples were held at
67.degree. C. for 4 minutes and then at 4.degree. C. until further
analysis. The PCR products were then cloned into the pGEM-T Easy
vector (Promega Corp. Madison, Wis.). Clones were sequenced for
verification.
Example 3
Expression Data Using Promoter Sequences
Promoter::GUS fusion constructs were prepared by the methods
described below. All vectors were constructed using standard
molecular biology techniques (Sambrook et al., Supra). A reporter
gene and a selectable marker gene for gene expression and selection
was inserted between the multiple cloning sites of the pBluescript
cloning vector (Stratagene Inc., 11011 N. Torrey Pines Rd., La
Jolla, Calif.). The ampicillin resistance gene was replaced with a
kanamycin resistance gene to allow use in bombardment experiments.
The reporter gene was the .beta.-glucuronidase (GUS) gene
(Jefferson, R. A. et al., 1986, Proc. Natl. Acad. Sci. (USA)
83:8447 8451) into whose coding region was inserted the second
intron from the potato ST-LS1 gene (Vancanneyt et al., Mol. Gen.
Genet. 220:245 250, 1990), to produce GUSINT, in order to prevent
expression of the gene in Agrobacterium (see Ohta, S. et al., 1990,
Plant Cell Physiol. 31(6):805 813. A fragment containing bases 2 to
310 from the terminator of the potato proteinase inhibitor (pinII)
gene (An et al., Plant Cell 1:115 122, 1989) was blunt-end ligated
downstream of the GUS coding sequence, to create the GUS expression
cassette. The 3' end of the terminator carried a NotI restriction
site. The respective promoter regions were ligated in frame to the
NcoI site 5' to the GUS gene at the start codon.
Jip1::GUS::pinII was constructed using the above plasmid digested
with NcoI, and PstI to provide insertion sites for the promoter.
The plasmid with the isolated Jip1 promoter in the pGEM-T Easy
cloning vector was digested with NcoI, and PstI. The fragment was
ligated into the digested expression cassette and successful
subcloning was confirmed by restriction analysis.
mi1ps3::GUS::pinII was constructed using the GUSINT::pinII
cassette. The cassette was digested with NcoI and HindIII. The
mi1ps3 promoter contained in the pGEM-T Easy cloning vector was
isolated through digestion with NcoI and HindIII. The fragment was
ligated into the digested expression cassette and successful
subcloning was confirmed by restriction analysis.
Lec1::GUS::Lec1 was prepared using the GUS::pinII cassette digested
digested with NcoI/HindIII, which had been modified to have a BgIII
site at the 3' end of the GUSINT coding region. The Lec1 promoter
in pGEM-T Easy was digested with NcoI and HindIII. The fragment was
then ligated into the vector and confirmed by restriction analysis
and sequencing. The Lec1 terminator was digested with BgIII/EcoRI
and ligated into the Lec1::GUS::PINII plasmid digested with
BgIII/EcoRI. Successful subcloning was confirmed by restriction
analysis.
The Agrobacterium transformation plasmids were constructed by
inserting the GUS expression cassettes as BstEII fragments into a
descendent plasmid of pSB11 which contained the BAR expression
cassette. Both the GUS, and BAR expression cassettes were located
between the right and left T-DNA. The GUS cassette was inserted
proximal to the right T-DNA border. The plasmid pSB11 was obtained
from Japan Tobacco Inc. (Tokyo, Japan). The construction of pSB11
from pSB21 and the construction of pSB21 from starting vectors is
described by Komari et al. (1996, Plant J. 10:165 174). The T-DNA
of the plasmid was integrated into the superbinary plasmid pSB1
(Saito et al., EP 672 752 A1) by homologous recombination between
the two plasmids. The plasmid pSB1 was also obtained from Japan
Tobacco Inc.
Competent cells of the Agrobacterium strain LBA4404 harboring pSB1
were created using the protocol as described by Lin (1995) in
Methods in Molecular Biology, ed. Nickoloff, J. A. (Humana Press,
Totowa, N.J.) The plasmid containing the expression cassettes was
electroporated into competent cells of the Agrobacterium strain
LBA4404 harboring pSB1 to create the cointegrate plasmid in
Agrobacterium using a BIO-RAD Micropulser (Cat# 165-2100, Hercules,
Calif.). Electroporation was performed by mixing 1 ul of plasmid
DNA (.about.100 ng) with 20 .mu.l of competent Agrobacterium cells
in a 0.2 cm electrode gap cuvette (Cat# 165-2086, BIO-RAD,
Hercules, Calif.). Electroporation was performed using the EC2
setting, which delivers 2.5 kV to the cells. Successful
recombination was verified by restriction analysis of the plasmid
after transformation of the cointegrate plasmid back into E. coli
DH5a cells.
Example 4
Transformation and Regeneration of Maize Callus via Agrobacterium
Preparation of Agrobacterium suspension
Agrobacterium was streaked out from a -80.degree. frozen aliquot
onto a plate containing PHI-L medium and cultured at 28.degree. C.
in the dark for 3 days. PHI-L media comprises 25 ml/l Stock
Solution A, 25 ml/l Stock Solution B, 450.9 ml/l Stock Solution C
and spectinomycin (Sigma Chemicals) added to a concentration of 50
mg/l in sterile ddH2O (stock solution A: K2HPO4 60.0 g/l, NaH2PO4
20.0 g/l, adjust pH to 7.0 w/KOH and autoclaved; stock solution B:
NH4Cl 20.0 g/l, MgSO4.7H2O 6.0 g/l, KCl 3.0 g/l, CaCl2 0.20 g/l,
FeSO4.7H2O 50.0 mg/l, autoclaved; stock solution C: glucose 5.56
g/l, agar 16.67 g/l (#A-7049, Sigma Chemicals, St. Louis, Mo.) and
autoclaved).
The plate can be stored at 4.degree. C. and used usually for about
1 month. A single colony was picked from the master plate and
streaked onto a plate containing PHI-M medium [yeast extract
(Difco) 5.0 g/l; peptone (Difco) 10.0 g/l; NaCl 5.0 g/l; agar
(Difco) 15.0 g/l; pH 6.8, containing 50 mg/L spectinomycin] and
incubated at 28.degree. C. in the dark for 2 days.
Five ml of either PHI-A, [CHU(N6) basal salts (Sigma C-1416) 4.0
g/l, Eriksson's vitamin mix (1000.times., Sigma-1511) 1.0 ml/l;
thiamine.HCl 0.5 mg/l (Sigma); 2,4-dichlorophenoxyacetic acid
(2,4-D, Sigma) 1.5 mg/l; L-proline (Sigma) 0.69 g/l; sucrose
(Mallinckrodt) 68.5 g/l; glucose (Mallinckrodt) 36.0 g/l; pH 5.2]
for the PHI basic medium system, or PHI-I [MS salts (GIBCO BRL) 4.3
g/l; nicotinic acid (Sigma) 0.5 mg/l; pyridoxine.HCl (Sigma) 0.5
mg/l; thiamine.HCl 1.0 mg/l; myo-inositol (Sigma) 0.10 g/l; vitamin
assay casamino acids (Difco Lab) 1 g/l; 2,4-D 1.5 mg/l; sucrose
68.50 g/l; glucose 36.0 g/l; adjust pH to 5.2 w/KOH and
filter-sterilize] for the PHI combined medium system and 5 ml of
100 mM (3' 5'-Dimethoxy-4'-hydroxyacetophenone, Aldrich chemicals)
were added to a 14 ml Falcon tube in a hood. About 3 full loops (5
mm loop size) Agrobacterium was collected from the plate and
suspended in the tube, then the tube vortexed to make an even
suspension. One ml of the suspension was transferred to a
spectrophotometer tube and the OD of the suspension adjusted to
0.72 at 550 nm by adding either more Agrobacterium or more of the
same suspension medium, for an Agrobacterium concentration of
approximately 0.5.times.109 cfu/ml to 1.times.109 cfu/ml. The final
Agrobacterium suspension was aliquoted into 2 ml microcentrifuge
tubes, each containing 1 ml of the suspension. The suspensions were
then used as soon as possible.
Embryo Isolation, Infection and Co-Cultivation:
About 2 ml of the same medium (here PHI-A or PHI-I) used for the
Agrobacterium suspension were added into a 2 ml microcentrifuge
tube. Immature embryos were isolated from a sterilized ear with a
sterile spatula (Baxter Scientific Products S 1565) and dropped
directly into the medium in the tube. A total of about 100 embryos
were placed in the tube. The optimal size of the embryos was about
1.0 1.2 mm. The cap was then closed on the tube and the tube
vortexed with a Vortex Mixer (Baxter Scientific Products S8223-1)
for 5 sec. at maximum speed. The medium was removed and 2 ml of
fresh medium were added and the vortexing repeated. All of the
medium was drawn off and 1 ml of Agrobacterium suspension added to
the embryos and the tube vortexed for 30 sec. The tube was allowed
to stand for 5 min. in the hood. The suspension of Agrobacterium
and embryos was poured into a Petri plate containing either PHI-B
medium [CHU(N6) basal salts (Sigma C-1416) 4.0 g/l; Eriksson's
vitamin mix (1000.times., Sigma-1511) 1.0 ml/l; thiamine.HCl 0.5
mg/l; 2.4-D 1.5 mg/l; L-proline 0.69 g/l; silver nitrate 0.85 mg/l;
gelrite (Sigma) 3.0 g/l; sucrose 30.0 g/l; acetosyringone 100 mM;
pH 5.8], for the PHI basic medium system, or PHI-J medium [MS Salts
4.3 g/l; nicotinic acid 0.50 mg/l; pyridoxine HCl 0.50 mg/l;
thiamine.HCl 1.0 mg/l; myo-inositol 100.0 mg/l; 2,4-D 1.5 mg/l;
sucrose 20.0 g/l; glucose 10.0 g/l; L-proline 0.70 g/l; MES (Sigma)
0.50 g/l; 8.0 g/l agar (Sigma A-7049, purified) and 100 mM
acetosyringone with a final pH of 5.8 for the PHI combined medium
system. Any embryos left in the tube were transferred to the plate
using a sterile spatula. The Agrobacterium suspension was drawn off
and the embryos placed axis side down on the media. The plate was
sealed with Parafilm tape or Pylon Vegetative Combine Tape (product
named "E.G.CUT" and is available in 18 mm.times.50 m sections;
Kyowa Ltd., Japan) and incubated in the dark at 23 25.degree. C.
for about 3 days of co-cultivation.
Resting, Selection and Regeneration Steps:
For the resting step, all of the embryos were transferred to a new
plate containing PHI-C medium [CHU(N6) basal salts (Sigma C-1416)
4.0 g/l; Eriksson's vitamin mix (1000.times. Sigma-1511) 1.0 ml/l;
thiamine.HCl 0.5 mg/l; 2.4-D 1.5 mg/l; L-proline 0.69 g/l; sucrose
30.0 g/l; MES buffer (Sigma) 0.5 g/l; agar (Sigma A-7049, purified)
8.0 g/l; silver nitrate 0.85 mg/l; carbenicillin 100 mg/l; pH 5.8].
The plate was sealed with Parafilm or Pylon tape and incubated in
the dark at 28.degree. C. for 3 5 days.
Longer co-cultivation periods may compensate for the absence of a
resting step since the resting step, like the co-cultivation step,
provides a period of time for the embryo to be cultured in the
absence of a selective agent. Those of ordinary skill in the art
can readily test combinations of co-cultivation and resting times
to optimize or improve the transformation
For selection, all of the embryos were then transferred from the
PHI-C medium to new plates containing PHI-D medium, as a selection
medium, [CHU(N6) basal salts (SIGMA C-1416) 4.0 g/l; Eriksson's
vitamin mix (1000.times., Sigma-1511) 1.0 ml/l; thiamine.HCl 0.5
mg/l; 2.4-D 1.5 mg/l; L-proline 0.69 g/l; sucrose 30.0 g/l; MES
buffer 0.5 g/l; agar (Sigma A-7049, purified) 8.0 g/l; silver
nitrate 0.85 mg/l; carbenicillin (ICN, Costa Mesa, Calif.) 100
mg/l; bialaphos (Meiji Seika K. K., Tokyo, Japan) 1.5 mg/l for the
first two weeks followed by 3 mg/l for the remainder of the time;
pH 5.8] putting about 20 embryos onto each plate.
The plates were sealed as described above and incubated in the dark
at 28.degree. C. for the first two weeks of selection. The embryos
were transferred to fresh selection medium at two-week intervals.
The tissue was subcultured by transferring to fresh selection
medium for a total of about 2 months. The herbicide-resistant calli
were then "bulked up" by growing on the same medium for another two
weeks until the diameter of the calli was about 1.5 2 cm.
For regeneration, the calli were then cultured on PHI-E medium [MS
salts 4.3 g/l; myo-inositol 0.1 g/l; nicotinic acid 0.5 mg/l,
thiamine.HCl 0.1 mg/l, Pyridoxine.HCl 0.5 mg/l, Glycine 2.0 mg/l,
Zeatin 0.5 mg/l, sucrose 60.0 g/l, Agar (Sigma, A-7049) 8.0 g/l,
Indoleacetic acid (IAA, Sigma) 1.0 mg/l, Abscisic acid (ABA, Sigma)
0.1 mM, Bialaphos 3 mg/l, carbenicillin 100 mg/l adjusted to pH
5.6] in the dark at 28.degree. C. for 1 3 weeks to allow somatic
embryos to mature. The calli were then cultured on PHI-F medium (MS
salts 4.3 g/l; myo-inositol 0.1 g/l; Thiamine.HCl 0.1 mg/l,
Pyridoxine.HCl 0.5 mg/l, Glycine 2.0 mg/l, nicotinic acid 0.5 mg/l;
sucrose 40.0 g/l; gelrite 1.5 g/l; pH 5.6] at 25.degree. C. under a
daylight schedule of 16 hrs. light (270 uE m-2sec-1) and 8 hrs.
dark until shoots and roots developed. Each small plantlet was then
transferred to a 25.times.150 mm tube containing PHI-F medium and
grown under the same conditions for approximately another week. The
plants were transplanted to pots with soil mixture in a greenhouse.
GUS+ events are determined at the callus stage or regenerated plant
stage.
For Hi-II, an optimized protocol was: 0.5.times.109 cfu/ml
Agrobacterium, a 3 5 day resting step, and no AgNO3 in the
infection medium (PHI-A medium).
Example 5
Transformation of Maize by Particle Bombardment
The inventive polynucleotides contained within a vector were
transformed into embryogenic maize callus by particle bombardment.
Transgenic maize plants were produced by bombardment of
embryogenically responsive immature embryos with tungsten particles
associated with DNA plasmid. The plasmid consisted of a selectable
and an unselectable marker gene.
Preparation of Particles
Fifteen mg of tungsten particles (General Electric), 0.5 to
1.8.mu., preferably 1 to 1.8.mu., and most preferably 1.mu., were
added to 2 ml of concentrated nitric acid. This suspension was
sonicated at 0.degree. C. for 20 minutes (Branson Sonifier Model
450, 40% output, constant duty cycle). Tungsten particles were
pelleted by centrifugation at 10000 rpm (Biofuge) for one minute,
and the supernatant removed. Two milliliters of sterile distilled
water were added to the pellet, and brief sonication used to
resuspend the particles. The suspension was pelleted, one
milliliter of absolute ethanol added to the pellet, and brief
sonication used to resuspend the particles. Rinsing, pelleting, and
resuspending of the particles was performed two more times with
sterile distilled water, and finally the particles resuspended in
two milliliters of sterile distilled water. The particles were
subdivided into 250-ml aliquots and stored frozen.
Preparation of Particle-Plasmid DNA Association
The stock of tungsten particles was sonicated briefly in a water
bath sonicator (Branson Sonifier Model 450, 20% output, constant
duty cycle) and 50 ml transferred to a microfuge tube. Equimolar
amounts of plasmid DNA are added to the particles for a final DNA
amount of 0.1 to 10 mg in 10 ml total volume, and briefly
sonicated: 1 mg total DNA was used. Fifty microliters of sterile
aqueous 2.5 M CaCl.sub.2 were added, and the mixture briefly
sonicated and vortexed. Twenty microliters of sterile aqueous 0.1 M
spermidine were added and the mixture briefly sonicated and
vortexed. The mixture was incubated at room temperature for 20
minutes with intermittent brief sonication. The particle suspension
was centrifuged, and the supernatant removed. Two hundred fifty
microliters of absolute ethanol were added to the pellet, followed
by brief sonication. The suspension was pelleted, the supernatant
removed, and 60 ml of absolute ethanol added. The suspension was
sonicated briefly before loading the particle-DNA agglomeration
onto macrocarriers.
Preparation of Tissue
Immature embryos of maize variety High Type II were the target for
particle bombardment-mediated transformation. This genotype is the
F1 of two purebred genetic lines, parents A and B, derived from the
cross of two know maize inbreds, A188 and B73. Both parents were
selected for high competence of somatic embryogenesis, according to
Armstrong et al., Maize Genetics Coop. News 65:92 (1991).
Ears from F1 plants were selfed or sibbed, and embryos aseptically
dissected from developing caryopses when the scutellum first became
opaque. This stage occurs about 9 13 days post-pollination, and
most generally about 10 days post-pollination, depending on growth
conditions. The embryos were about 0.75 to 1.5 millimeters long.
Ears were surface sterilized with 20 50% Clorox for 30 minutes,
followed by three rinses with sterile distilled water.
Immature embryos were cultured with the scutellum oriented upward,
on embryogenic induction medium comprised of N6 basal salts,
Eriksson vitamins, 0.5 mg/l thiamine HCl, 30 gm/l sucrose, 2.88
gm/l L-proline, 1 mg/l 2,4-dichlorophenoxyacetic acid, 2 gm/l
Gelrite, and 8.5 mg/l AgNO3. Chu et al., Sci. Sin. 18:659 (1975);
Eriksson, Physiol. Plant 18:976 (1965).
The medium was sterilized by autoclaving at 121.degree. C. for 15
minutes and dispensed into 100.times.25 mm Petri dishes. AgNO3 was
filter-sterilized and added to the medium after autoclaving. The
tissues were cultured in complete darkness at 28.degree. C. After
about 3 to 7 days, most usually about 4 days, the scutellum of the
embryo swelled to about double its original size and the
protuberances at the coleorhizal surface of the scutellum indicated
the inception of embryogenic tissue. Up to 100% of the embryos
displayed this response, but most commonly, the embryogenic
response frequency was about 80%.
When the embryogenic response was observed, the embryos were
transferred to a medium comprised of induction medium modified to
contain 120 gm/l sucrose. The embryos were oriented with the
coleorhizal pole--the embryogenically responsive tissue--upwards
from the culture medium. Ten embryos per Petri dish were located in
the center of a Petri dish in an area about 2 cm in diameter. The
embryos were maintained on this medium for 3 16 hour, preferably 4
hours, in complete darkness at 28.degree. C. just prior to
bombardment with particles associated with plasmid DNA containing
the selectable and unselectable marker genes.
To effect particle bombardment of embryos, the particle-DNA
agglomerates were accelerated using a DuPont PDS-1000 particle
acceleration device. The particle-DNA agglomeration was briefly
sonicated and 10 ml were deposited on macrocarriers and the ethanol
allowed to evaporate. The macrocarrier was accelerated onto a
stainless-steel stopping screen by the rupture of a polymer
diaphragm (rupture disk). Rupture is effected by pressurized
helium. The velocity of particle-DNA acceleration is determined
based on the rupture disk breaking pressure. Rupture disk pressures
of 200 to 1800 psi were used. Multiple disks were used to effect a
range of rupture pressures.
The shelf containing the plate with embryos was placed 5.1 cm below
the bottom of the macrocarrier platform (shelf #3). To effect
particle bombardment of cultured immature embryos, a rupture disk
and a macrocarrier with dried particle-DNA agglomerates were
installed in the device. The He pressure delivered to the device
was adjusted to 200 psi above the rupture disk breaking pressure. A
Petri dish with the target embryos was placed into the vacuum
chamber and located in the projected path of accelerated particles.
A vacuum was created in the chamber, preferably about 28 in Hg.
After operation of the device, the vacuum was released and the
Petri dish removed.
Bombarded embryos remain on the osmotically-adjusted medium during
bombardment, and 1 to 4 days subsequently. The embryos are
transferred to selection medium comprised of N6 basal salts,
Eriksson vitamins, 0.5 mg/1 thiamine HCl, 30 gm/l sucrose, 1 mg/l
2,4-dichlorophenoxyacetic acid, 2 gm/l Gelrite, 0.85 mg/l Ag NO3
and 3 mg/l bialaphos (Herbiace, Meiji). Bialaphos was added
filter-sterilized. The embryos were subcultured to fresh selection
medium at 10 to 14 day intervals. After about 7 weeks, embryogenic
tissue, putatively transformed for both selectable and unselected
marker genes, proliferated from the bombarded embryos. Putative
transgenic tissue was rescued, and that tissue derived from
individual embryos was considered to be an event and propagated
independently on selection medium. Two cycles of clonal propagation
were achieved by visual selection for the smallest contiguous
fragments of organized embryogenic tissue.
For regeneration of transgenic plants, embryogenic tissue was
subcultured to a medium comprising MS salts and vitamins (Murashige
& Skoog, Physiol. Plant 15: 473 (1962)), 100 mg/l myo-inositol,
60 gm/l sucrose, 3 gm/l Gelrite, 0.5 mg/l zeatin, 1 mg/l
indole-3-acetic acid, 26.4 ng/l cis-trans-abscissic acid, and 3
mg/l bialaphos in 100.times.25 mm Petri dishes, and incubated in
darkness at 28.degree. C. until the development of well-formed,
matured somatic embryos could be seen. This required about 14 days.
Well-formed somatic embryos were opaque and cream-colored, and
comprised of an identifiable scutellum and coleoptile. The embryos
were individually subcultured to a germination medium comprising MS
salts and vitamins, 100 mg/l myo-inositol, 40 gm/l sucrose and 1.5
gm/l Gelrite in 100.times.25 mm Petri dishes and incubated under a
16 hour light::8 hour dark photoperiod and 40 meinsteinsm-2sec-1
from cool-white fluorescent tubes. After about 7 days, the somatic
embryos germinated and produced a well-defined shoot and root. The
individual plants were subcultured to germination medium in
125.times.25 mm glass tubes to allow further plant development. The
plants were maintained under a 16 hour light:8 hour dark
photoperiod and 40 meinsteinsm-2sec-1 from cool-white fluorescent
tubes. After about 7 days, the plants were well-established and
transplanted to horticultural soil, hardened off, and potted into
commercial greenhouse soil mixture and grown to sexual maturity in
a greenhouse.
Example 6
Northern Analysis of Gene Expression in Vegetative Tissue and
Developing Kernels
Total RNA (10 g) was size fractionated on a 1% formaldehyde agarose
gel and transferred to a nitrocellulose membrane. In separate
experiments, membranes were hybridized under stringent conditions
with 32P-labelled probes representing cDNA fragments of Jip1 and
Lec1. After extensive washing to remove unbound probe, membranes
were exposed on X-ray film. RNA samples were obtained from
vegetative tissues (Tassel, leaf, primary root) as well as
developing maize kernels.
The Jip1 expression pattern showed no expression in vegetative
tissues or 0 5 DAP whole kernel. Jip1 was predominantly expressed
in 15 40 DAP embryo with some weaker expression in the endosperm
and pericarp.
The Lec 1 expression pattern showed no expression in vegetative
tissues or endosperm tissues. Lec1 was predominately expressed in
10 DAP embryo tissue with weaker expression in 15 DAP embryo.
Expression was off by 20 DAP in the embryo.
All publications and patent applications mentioned in the
specification are indicative of the level of those skilled in the
art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
Although the foregoing invention has been described in some detail
by way of illustration and example for purposes of clarity of
understanding, it will be obvious that certain changes and
modifications can be practiced within the scope of the appended
claims.
SEQUENCE LISTINGS
1
1911247DNAZea maizepromoter(1)...(1247) 1atcgtacata aagttggatt
atacaatatc catttacaac taaatagaaa ccaattaatt 60taaaaaacta aaaaaacctt
tatcaccgta caggagaaga gagcatcaac ttgctattag 120ttttatgcat
ttaaacaccc ttcgaaccat cagcagtggt tgataggttt aactgatact
180aatatcttgt ctttaatact agcaccaact gataataatc tttcgaacac
atgttattat 240ctattgttga ctttaatcaa tactaaatcc aagatattag
tagagatgtt agtatagatt 300aaggtgatgt ttgaatgcac tagagctaat
agttagtagc taaaattagt tggagacatt 360caaacaccct atcaattatt
agttattttt agtaaattag ttaatagtta gttagttatt 420tataagctag
ctttttttac tagcaatttt ttagccaact aacaattagt tttagtgtat
480tcaaataccc ctaagccgtt aagtgatgct ctttctagaa tcttaaccgt
atgtggagac 540aacattttca taggtgtact gtttaagtca ccgtcagtga
taataatatt ttcacatgcg 600gtttcttaag caaaccgcca gtgctaatga
tatttacact agcgggctgc taaagaaaac 660cgcccgtgct aaagatattt
acactagcgg ttggtgaaca actgcctgtg aaaaaagccg 720attcctacta
gcccctagct tgcactggcg acataaaaaa cgtcagtgaa aatagctcta
780ggatcgtcac tatagagctt ctatgtactt agtggttaga actgatattg
tagtgcacca 840agtgccgatt ttaattaaac caatactaaa tactagtaaa
taatactagt ggtctgaatt 900cgatttctat agtaatgttt gcttgcaagc
cgcaaataga gtaaacattc gtcgtcacag 960aaatccacat tacatcaagg
tccatggcgg ccggccacgt acccatccca cgcgtcgctg 1020cggaggacac
gtgttggctg accggacagt tggccgatca gacagtggac agaccggaca
1080atagaagaag aagacgacga cggcggcggc accgccgagt aggtgcatgg
tcacgctagc 1140tgtagctttt tgcagagcgt cgtctgtaaa tacgtagccc
ttccacaagc gaggcaaggg 1200gggagagagt atcgtcagct agcagagaga
gtgcgtagca actagca 1247226DNAArtificial SequenceJip1 forward primer
2tcgggagaag tcactagcgg ggaact 26327DNAArtificial
Sequenceprimer_bind(1)...(27)Jip1 nested forward primer 3gagcagggtt
ctccgccatt gctagtt 274752DNAZea mayspromoter(1)...(752) 4aagctttgat
caaaagcgcc cgccacttct aaaggtcagg ggtcttgcgt tctgcccctc 60gtgcttcctt
caaattctgg acctagtgga tcaatttacg tacacctcag caaccgatgc
120agccagtaag tatgatgagc acgattgtga cgtgttgggg tcatggtcaa
tggcaaccga 180gcacgaattg gtagtgtcag ctttttgtac acgtgatagc
atttgattcg ttcattcaat 240ttgaactgtt tgaacttatg tatagagaaa
ttagtccaac tcatgtttaa taaatagtat 300aaaacccatc gaatttctga
attatgatag caggtatcca ttgtcatcgc tcagtcacag 360aggcagccac
tgccgacggg cgacggccga cggcctccca tttcgatccc ctcctactcc
420tatgctgcgg tccagcataa gttcgggact tccggcaatc cgccggcgcc
cgtcggctca 480aatcgcatct accgcggcta gaagctctct cttcctccct
ccgatccggt ggggtccatt 540tccttcaatt gtggcagtgg ccgtctcgaa
ccctctataa atcccccacc ccggacaccc 600ttccccgacc acacggtcca
cacagcccaa caaaggagcg cggcggcccc tccttccttc 660ctcccacttc
tctcgcgcgg cgctcgctta cctcgcctcg cattccgttc gagcagggga
720gcggcagtga gaagggaggg aattaaggca cc 752530DNAArtificial
Sequenceprimer_bind(1)...(30)Mi1ps3 forward primer 5cggaagctct
cgatgaacat cttgccttaa 3061433DNAZea mayspromoter(1)...(1433)
6tcctaatctt caaataacca tctcaaaagt tttttaaaac atcttttgag gatatgtatc
60ccatagccct agagcgctaa attgactact tttagtcgat taaaaggtat tagacatcct
120tacaagtcct aagtatcaaa tcaccttcta tcggctatac acaactaacg
gaagttatct 180ctagtcacac taacttatgt cggtttccgc atggcagatc
aaaattagct aacttttgtt 240ggctaataag agcaattcca aaagaacgtg
taaactaatc tcaaaacaga tattagttaa 300gaatagtaat ttttcttact
ccaacagttc cctcagtctt ccccaaaaaa ttaagcgttc 360cgcatccaca
gcctcctctc ggtcgtattt tggtgtgttt catccctccc caatccattt
420ctcaacgtat cagatcatcc accgcctacg acgactgtac agtttgcgtc
acatatcaca 480tttaaaggaa ctgttggagt acccatcata attcactctt
aaaaaatttt agcctgctct 540caataatcaa ttgggggggt aaaattttta
acatcctttc ggatctaatc caacttatgg 600aagttagcta gctctggtcg
cgctaacttc tgtcgatcgc ctattagcta atactccatc 660tgtcccatta
tataaggtat aaccaactct gattcaaaga ccaaaaatat acttaattgt
720gtctatacca cttcatcgat gtacgtatgc atagaaagag cacatcttat
attgtggaac 780aagaacaaaa atatggttac gccttatatt ataagacgta
gaaatcaatg gtttacaata 840gccaagaata gatgttttta tttatttcct
atatagatgt ttttatttat ttcctatatg 900tttcacaata gccttatatt
gtgccgaaaa tttaggcaca cgtgccacga acgtctgaaa 960tgtactccgc
gcgtattacc atgcactacg acgtacgtag gagtatgtac gttgaaccaa
1020gcacacatat atctctgaca cagtacaatg atatactaca acaacaacag
tactgcccaa 1080ttcatccatt ttcacgttcc atcttccgcg tgtgacaact
cgatcggcca cgcacgcaga 1140cgacgacgga gcagtacttc acagaatcct
ccgccactcg tcacaccaac aggcgcgcgc 1200tggtgcgcat gcatcatgtg
catgccatcg tccgtccctt ggcgtgcctc ggtagacggt 1260agctagagta
gtagcctgtg cttgctaccc ctggtcaaca catcgtagcc tcctatattt
1320aacgtatcct cacacatcac aagaacgaca cacagaaacc agtagccact
actccatcca 1380ccacgagcga gcgagcgata accctagcta gcttcaggat
ccagcgagag ccc 1433720DNAArtificial
Sequenceprimer_bind(1)...(20)Lec 1 prom. forward probe 7acaccacgag
cgcgcgataa 20820DNAArtificial Sequenceprimer_bind(1)...(20)Lec 1
prom. reverse probe 8tggtggtggt gcggtagcat 209695DNAZea
maysterminator(1)...(695) 9tacctagttc gtacgtcgtt cgacttgagc
aagccatcga tctgctgatc tgaacgtacg 60ctgtattgta cacgcatgca cgtacgtatc
ggcggctagc tctcctgttt aagttgtact 120gtgattctgt cccggccggc
tagcaactta gtatcttcct tcagtctcta gtttcttagc 180agtcgtagaa
gtgttcaatg cttgccagtg tgttgtttta gggccggggt aaaccatccg
240atgagattat ttcatgcacg ctttttagac tgacgactgt tcgtgtgtgt
cttctgcgca 300gttccgctcc tagcacaatt atatcatcct tgatgatcgt
ttaacgcaac agtcttctct 360ggaggtctag acctagcgga tcttttgttg
tactcccttc tatacgtaca tgcatactac 420acgtacgtac ggcggcggta
cggcagctac atattcgtcg ttcgagtgtg atgcatgggt 480tgtctttaaa
gcccttcgtc gttctagctg ctggcctgct gctatagcct gtaggtgagg
540tgttcgtggc ttgcgacgcg cgggaggaag cacgacggac ggtggcggcg
catgttcttg 600actctggact attgcgcgta ggcgcgtgac caagttccac
ggtggcgtgc gcggctgcag 660cgaggcgccc gcgacaagtg ccggcgatcc cgcag
6951045DNAArtificial Sequenceprimer_bind(1)...(45)Lec1 term.
forward primer 10gtcgacagat ctgttaacct agttcgtacg tcgttcgact tgagc
451136DNAArtificial Sequenceprimer_bind(1)...(36)Lec1 term. reverse
primer 11gggccccgtg cggcaacaaa aatagacctg acctca
361229DNAArtificial Sequenceprimer_bind(1)...(29)Jip1 forward
nested primer 12cctttatcac cgtacaggag aagagagca 291326DNAArtificial
Sequenceprimer_bind(1)...(26)Jip1 reverse nested primer
13tattgtccgg tctgtccact gtctga 261428DNAArtificial
Sequenceprimer_bind(1)...(28)Mi1ps3 nested forward primer
14gagctcctcc acgcgatcaa aagcgccc 281537DNAArtificial
Sequenceprimer_bind(1)...(37)Mi1ps3 nested reverse primer
15cccgggccat ggtcatgcct taattccctc ccttctc 371629DNAArtificial
Sequenceprimer_bind(1)...(29)Lec1 promoter forward nested primer
16tcgaggtcga cggtatcgat aagcttcct 291749DNAArtificial
Sequenceprimer_bind(1)...(49)Lec1 nested reverse primer
17gtcgacccat gggctctcgc tggatcctga agctagctag ggttatcgc
491845DNAArtificial Sequenceprimer_bind(1)...(45)Lec1 term. forward
nested primer 18gtcgacagat ctgttaacct agttcgtacg tcgttcgact tgagc
451936DNAArtificial Sequenceprimer_bind(1)...(36)Lec1 term. reverse
nested primer 19gggccccgtg cggcaacaaa aatagacctg acctca 36
* * * * *